Purification and characterization of extracellular α-amylase from a thermophilic <i>Anoxybacillus thermarum</i> A4 strain

Baltas, Nimet; Dincer, Barbaros; Ekinci, Arife Pinar; Kolayli, Sevgi; Adiguzel, Ahmet

doi:10.1590/1678-4324-2016160346

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Engineering, Technology and Techniques • Braz. arch. biol. technol. 59 • Jan-Dec 2016 • https://doi.org/10.1590/1678-4324-2016160346 copy

Purification and characterization of extracellular α-amylase from a thermophilic Anoxybacillus thermarum A4 strain

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

α-Amylase from Anoxybacillus thermarum A4 was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration chromatography, with 29.8-fold purification and 74.6% yield. A4 amylase showed best performance for soluble potato starch hydrolysis at 70 °C and pH 5.5-10.5. A4 amylase was extremely stable at +4 °C, and the enzyme retained over 65% of its original α-amylase activity at 70 °C and 43% at 90 °C. The enzyme's K_m values for soluble starch, amylopectin and amylose substrates were obtained as 0.9, 1.3 and 0.5 mg/mL, respectively. EDTA, Hg²⁺, B₄O₇ ^2-, OH^-, CN^- _, and urea exhibited different inhibition effects; their IC₅₀ values were identified as 8.0, 5.75, 16.5, 15.2, 8.2 and 10.9 mM, respectively. A4 amylase exhibited extreme stability toward some surfactants and perfect match for a wide variety of commercial solid and liquid detergents at 55 °C. So, it may be considered to be potential applications for detergent and other industrial uses.

Key words:
Anoxybacillus thermarum A4; Extracellular α-Amylase; Highly thermostable; Purification

Metal ions	Remaining activity (%)
Metal ions	1 mM	5 mM	10 mM	20 mM
None	100	100	100	100
Mn²⁺	132	148	156	175
Ca²⁺	120	129	134	141
Cu²⁺	121	124	125	132
Ba²⁺	102	106	112	130
Co²⁺	100	117	138	148
Zn²⁺	100	103	107	114
Fe²⁺	95.94	102	119	123
Ni²⁺	95.61	98.67	114	117
Mg²⁺	95.03	74.83	70.33	65.98
Hg²⁺	66.31	51.3	42.4	37.3
EDTA	82.9	64.8	43.1	27

Anions	Remaining activity (%)
Anions	1 mM	5 mM	10 mM	20 mM
None	100	100	100	100
ClO₄ ^-	100	100	100	100
Cl^-	100	100	100	100
Ac^-	100	100	100	100
NO₃ ^-	100	100	100	100
SO₄ ^-2	102	104	105	106
N₃ ^-	98.98	89.94	100	100
C₂O₄ ^-2	110	106	102	101
HCO₃ ^-	103	101	95.16	88.71
HPO₄ ^-2	100	97.66	94.27	90.97
HSO₃ ^-	99.58	97.90	95.96	89.11
WO₄ ^2-	98.77	95.24	93.46	83.22
C₈H₁₁N₂O₃ ^-	99.1	94.75	92.77	82.5
SO₃ ^-	99.19	97.18	85.40	71.53
CO₃ ^-2	98.56	92.82	81.37	60.48
PO₄ ^-3	96.4	80.56	72.58	58.62
B₄O₇ ^-2	97.1	81.93	61.69	43.54
OH^-	98	91.20	65.73	37.1
CN^-	97.75	73.79	40.16	5.65
N₂ H₄CO	93.9	67.2	52.1	23.41

Denaturing agents, various commercial solid and liquid detergents	Remaining activity (%)
	0.5 %	1 %	2 %
None	100	100	100
Triton X-114	113	114	115
Triton X-100	109	107	104
Tween-80	100	100	100
Tween-20	100	100	100
SDS	100	98	95.4
Liquid dishwashing machine detergent	100	100	100
Liquid hand dishwashing detergent	100	98	95.6
Liquid laundry detergent	100	99	97
Solid laundry detergent	98.3	95.8	92.7
Spot remover	100	98.2	96

Substrate	Activity (U)	Relative Activity (%)
Amylopectin	115.8	93
Amylose	140.8	113
Potato starch	124.9	100
Corn starch	108.6	86.9
Wheat starch	110.8	88.7
β-Cyclodextrin	-	-
Glycogen	62.6	50
Cellulose	-	-

	V_max (U/mg protein)	K_m (mg/mL)	k_cat (s^-1)	K_cat/K_M (mLs^-1mg^-1)
Potato starch	833.3±3.7	0.9±0.08	694.4	771.6
Amylose	769±2.4	0.5±0.04	640.8	1281.7
Amylopectin	1000±4.5	1.3±0.12	833.3	641.0

Instituto de Tecnologia do Paraná - Tecpar Rua Prof. Algacyr Munhoz Mader, 3775 - CIC, 81350-010 Curitiba PR Brazil, Tel.: +55 41 3316-3052/3054, Fax: +55 41 3346-2872 - Curitiba - PR - Brazil
E-mail: babt@tecpar.br

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[1] *Authors for correspondence: nimetaktas07@hotmail.com

	Total Volume (mL)	Total Protein (mg)	Total Activity (U)	Spesific activity (U/mg protein)	Purification fold	Yield (%)
Crude enzyme extract	9900	538.6	21780	40.4	1	100
Concentraion with Amicon filter after precipitate (40-80)% As^a	15	92.25	16375	177.5	4.4	75.2
Sephadex G-100	15	13.5	16250	1203.7	29.8	74.6